The present invention relates generally to compositions and methods for modulating the activity of the MAP/ERK kinase MEK3 and/or other MEK family members. The invention is more particularly related to TAO proteins, and variants thereof that stimulate phosphorylation and activation of MEK substrates, such as MEK3. The invention is further related to the use of such proteins, for example, to activate a stress-responsive MAP kinase pathway in an organism and to identify antibodies and other agents that inhibit or activate signal transduction via such a pathway.
MAP kinase pathways are conserved signal transduction pathways that activate transcription factors, translation factors and other target molecules in response to a variety of extracellular signals. Each pathway contains a MAP kinase module, consisting of a MAP kinase or ERK, a MAP/ERK kinase (MEK), and a MEK kinase (MEKK). In higher eucaryotes, activation of MAP kinase pathways has been correlated with cellular events such as proliferation, oncogenesis, development and differentiation. Accordingly, the ability to regulate signal transduction via these pathways could lead to the development of treatments and preventive therapies for human diseases associated with MAP kinase pathways, such as inflammatory diseases, autoimmune diseases and cancer.
Several MAP kinase pathways have been found in S. cerevisiae (Hunter and Plowman, Trends in Biochem. Sci. 22:18-22, 1997), and parallel mammalian pathways have been identified based upon sequences of mammalian ERKs and yeast MAP kinases, KSS1 and FUS3 (Boulton et al., Science 249:64-67, 1990; Courchesne et al., Cell 58: 1107-1119, 1989; Elion et al., Cell 60:649-664, 1990). The best delineated yeast MAP kinase pathway, activated by mating pheromones, is controlled by a receptor-G protein system, includes a Cdc42 small G protein, and requires at least three protein kinases, Ste20p (Leberer et al., EMBO J. 11:4815-4828, 1992; Ramer et al., Proc. Natl. Acad. Sci. USA 90:452-456, 1993), Ste11p (Rhodes et al., Genes Dev. 4:1862-1874, 1990), and Ste7p (Teague et al., i Proc. Natl. Acad Sci. USA 83:7371-7375, 1986), upstream of the MAP kinase Fus3p (Elion et al., Cell 60:649-664, 1990).
Ste20p was isolated from S. cerevisiae as a gene whose product functions downstream of the xcex2xcex3 subunits of a heterotrimeric G protein but upstream of enzymes in the MAP kinase module (MEKK, MEK, ERK) of the pheromone response pathway (Leberer et al., EMBO J. 11:4815-4828, 1992; Ramer et al., Proc. Natl. Acad. Sci. USA 90:452-456, 1993). Ste11p, the MEKK, may be one of the Ste20p substrates (Wu et al., J. Biol. Chem. 270:15984-15992, 1990); thus, Ste20-like enzymes may activate MEKKs in mammalian MAP kinase pathways. Ste20p, like its best studied mammalian counterparts, the p21-activated protein kinases (PAKs), is thought to be regulated by binding to Cdc42 through a conserved Cdc42/Rac interactive binding region, or CRIB domain (Burbelo et al., J. Biol. Chem. 270:29071-29074, 1995).
Mammalian relatives of Ste20p are diverse and include the PAK subfamily (PAK1,2,3) and the mixed lineage kinase (MLK) subfamily, including the dual leucine zipper kinase (DLK), germinal center kinase (GCK), and the Nck-interacting kinase, NIK. In the past year, newly identified Ste20p-related kinases include members of the MLK subfamily, SOK-1, Krs-1 and -2, and MUK. MUK was isolated in a screen for MEKK isoforms, but in fact shows more identity to MLK. In transfected cells several of these enzymes, as first shown with GCK, increase the activity of the stress-responsive kinases, particularly SAPK/JNK. In the case of NIK and GCK, they may work by binding to MEKK (Su et al., EMBO J. 16:1279-1290, 1997). However, several of these Ste20p-related enzymes also have MEKK activity. For example, DLK phosphorylates and potently activates MEKs that lie in the stress-responsive cascades.
Further characterization of members of these pathways, and the identification of additional members, is critical for understanding the signal transduction pathways involved and for developing methods for activating or inactivating MEKs and MAP kinase pathways in vivo. Accordingly, there is a need in the art for improved methods for modulating the activity of members of MAP kinase pathways, and for treating diseases associated with such pathways. The present invention fulfills these needs and further provides other related advantages.
Briefly stated, the present invention provides compositions and methods for modulating the activity of MAP/ERK kinases such as MEK3, and stress-responsive MAP kinase pathways. Within certain aspects, the present invention provides TAO polypeptides. Within one such aspect, the polypeptide may comprise an amino acid sequence provided in SEQ ID NO:2 or SEQ ID NO:4 or a variant thereof in which the ability to phosphorylate MEK3 is not substantially diminished. In certain embodiments, such a polypeptide may comprise a sequence that differs from a sequence recited in SEQ ID NO:2 or SEQ ID NO:4 only in conservative substitutions and/or modifications at no more than 10% of the amino acid residues. In certain other embodiments, the polypeptide may be a constitutively active variant.
Within other aspects, the present invention provides polypeptides comprising an amino acid sequence provided in SEQ ID NO:2 or SEQ ID NO:4 modified at no more than 10% of the amino acid residues, such that the polypeptide is rendered constitutively inactive.
Within further aspects, the present invention provides polypeptides capable of phosphorylating MEK3, wherein the polypeptide does not detectably phosphorylate MEK1 or MEK2.
The present invention further provides, within other aspects, isolated polynucleotides encoding polypeptides as described above. Isolated polynucleotides comprising one or more sequences recited in any one of SEQ ID NOs:5-16, or a variant thereof, wherein the polynucleotide encodes a polypeptide capable of phosphorylating MEK3, are also provided. Polypeptides encoded by such polynucleotides are further provided. Recombinant expression vectors comprising any of the above polynucleotides, and host cells transformed or transfected with such expression vectors, are provided within related aspects.
Within other aspects, the present invention provides antisense polynucleotides comprising at least 10 nucleotides complementary to a polynucleotide as described above.
Within further aspects, pharmaceutical compositions are provided, comprising: (a) a polypeptide or polynucleotide as described above; and (b) a physiologically acceptable carrier.
The present invention further provides, within other aspects, methods for phosphorylating a MEK3 polypeptide, comprising contacting a MEK3 polypeptide with a polypeptide according to claim as described above, thereby phosphorylating the MEK3 polypeptide.
Within further aspects, the present invention provides methods for activating a member of a stress-responsive MAP kinase pathway in an organism, comprising administering to an organism a polypeptide as described above, and thereby activating a member of a stress-responsive MAP kinase pathway.
Within other aspects, methods are provided for phosphorylating a MEK3 polypeptide comprising contacting a MEK3 polypeptide with a polypeptide as described above, and thereby phosphorylating the MEK3 polypeptide.
The present invention further provides methods for activating a member of a stress-responsive MAP kinase pathway in an organism, comprising administering to an organism a polypeptide as described above, and thereby activating a member of the stress-responsive MAP kinase pathway.
Within further aspects, the present invention provides methods for screening for an agent that modulates signal transduction via a stress-responsive MAP kinase pathway, comprising: (a) contacting a candidate agent with a polypeptide as described above; and (b) subsequently measuring the ability of the polypeptide to modulate the activity of a MEK3 polypeptide, and thereby evaluating the ability of the compound to modulate signal transduction via a stress-responsive MAP kinase pathway.
The present invention further provides, within other aspects, monoclonal antibodies and antigen-binding fragments thereof that specifically bind to a polypeptide as described above. Such monoclonal antibodies or fragments thereof may inhibit the phosphorylation of MEK3 by the polypeptide. Pharmaceutical compositions comprising: (a) an antibody or antigen-binding fragment thereof as described above; and (b) a physiologically acceptable carrier are also provided.
Within other aspects, the present invention provides methods for treating a patient afflicted with a disease associated with a stress-responsive MAP kinase pathway, comprising administering to a patient a compound that modulates the phosphorylation of MEK3. Within certain embodiments, the compound comprises a monoclonal antibody or antigen-binding fragment thereof or a nucleotide sequence. Within such methods, the compound may inhibit phosphorylation of MEK3 and the disease may be inflammation, an autoimmune disease, cancer or a degenerative disease. Alternatively, the compound may enhance the phosphorylation of MEK3 and the disease may be insulin-dependent diabetes or a neurodegenerative disease.
Within other aspects, methods are provided for determining the presence or absence of TAO kinase activity in a sample, comprising evaluating the ability of the sample to phosphorylate a MEK3 polypeptide, and thereby determining the presence or absence of TAO kinase activity in the sample.
Within related aspects, kits are provided for detecting TAO kinase activity in a sample, comprising a MEK3 polypeptide in combination with a suitable buffer.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.